EP1496111A2 - Variants of 3-Phosphoglycerate dehydrogenase with reduced inhibition by L-serine and genes encoding the same - Google Patents

Abstract

A 3-phosphoglyceraldehyde dehydrogenase (I) that, compared with the wild-type enzyme of Escherichia coli, has lower sensitivity to serine, is new, where it differs from the wild-type sequence (410 amino acids, given in the specification) by having 349Gly and/or 372Thr replaced by other amino acids. Independent claims are also included for the following: (1) the serA allele (II) that encodes (I), differing from the wild-type serA (1233 base pair (bp) sequence, given in the specification) by having codons for 349Gly and/or 372Thr replaced by codons for other amino acids; (2) a vector that contains (II) under control of a promoter; (3) method for preparing a microbial strain by introducing a vector of (2); (4) microbial strain, suitable for fermentative production of amino acids of the phosphoglycerate family, their derivatives or compounds, or of compounds derived from C1 metabolism, that contains (I); and (5) producing amino acids of the phosphoglycerate family, their derivatives or compounds, or compounds derived from C1 metabolism by culturing strains of (4).

Description

The invention relates to variants of 3-phosphoglycerate dehydrogenase with reduced inhibition by L-serine and coding for it Genes.

The production of the twenty natural, proteinogenic amino acids Nowadays, it is predominantly fermented by microorganisms accomplished. It exploits that microorganisms via appropriate biosynthetic pathways for synthesis of natural amino acids.

However, such biosynthetic pathways are subject to wild-type strains a strict control that ensures that the amino acids be made only for personal use of the cell. One For example, important control mechanism in many biosyntheses the phenomenon of feedback inhibition (or end product inhibition). Usually the enzyme of a biosynthetic pathway, this is the preliminary enzymatic reaction of this Biosynthesis pathways catalyzed by the end product of Biosyntheseweges inhibited. The inhibition usually takes place through an allosteric Binding of the final product to the enzyme instead of and causes a conformational change to an inactive state. This ensures that when an accumulation of the final product in the cell further synthesis by inhibition of introductory step is prevented.

An efficient industrial production of metabolic products (such as amino acids) is therefore only possible if the restrictions by feedback inhibition of a metabolic pathway can be repealed and thus microorganisms available which, in contrast to wild-type organisms, a drastically increased production capacity for production have the desired metabolite.

The phosphoglycerate family of amino acids is defined by that they are amino acids involved in their biosynthesis derived from the 3-phosphoglyceric acid. Of the natural path of metabolism leads to it over the intermediates 3-Phospohydroxypyruvat and 3-phospho-Lserin to L-serine. L-serine can continue to glycine or over O-acetyl-serine be converted to L-cysteine. Also L-tryptophan is to be counted to this group, since it is also in derived from the biosynthesis of L-serine. Likewise, unnatural Amino acids described in US 2002/0039767 A1 Process to be assigned to the phosphoglycerate family.

Compounds derived from the C1 metabolism show also a dependence on the biosynthesis of the amino acids the phosphoglycerate family. This is based on the fact that in the conversion of L-serine to glycine tetrahydrofolate acts as a C1 group acceptor and the loading tetrahydrofolate as a central methyl group donor in C1 metabolism in many biosyntheses (e.g., L-methionine, nucleotides, pantothenic acid, etc.). According to the invention, compounds derive from the C1 metabolism thus preferably Compounds that in their biosynthesis of a C1 group transfer depend on tetrahydrofolic acid.

The initial step in the biosynthesis of amino acids of the phosphoglycerate family is the oxidation of D-3-phosphoglyceric acid to 3-phosphohydroxypyruvate and is catalyzed by the enzyme 3-phosphoglycerate dehydrogenase (PGD) [EC 1.1.1.95]. NAD ⁺ , which is converted into NADH / H ⁺ , serves as the acceptor for the reduction equivalents formed during the reaction.

PGD enzymes are known from a variety of organisms (e.g. Rattus norvegicus, Arabidopsis thaliana, Escherichia coli, Bacillus subtilis). The better characterized microbial representatives of feedback inhibition by L-serine.

At the level of the amino acid sequence are the microbial PGD enzymes in the N-terminal part (amino acid 1-340 of the E. coli sequence) very similar to each other, whereas the C-terminal portion has little similarity. Especially in this C-terminal Part is localized to the regulatory domain, the is responsible for serine inhibition (Peters-Wendisch et al., 2002, Appl. Microbiol. Biotechnol. 60: 437-441).

The best characterized PGD is that of Escherichia coli. The enzyme has been extensively biochemically studied (Dubrow & Pizer, 1977, J. Biol. Chem. 252, 1527-1538) and is subject to allosteric feedback inhibition by L-serine with an inhibitor constant K _i of 5 μM.

This feedback inhibition stands for an efficient production of Amino acids of the phosphoglycerate family in the way and was therefore already the goal of molecular biological approaches.

Thus, in the document EP0620853A variants of Escherichia coli PGD with reduced sensitivity to inhibition by Serine described a modification in the C-terminal 25% of the wild-type PGD (i.e., amino acids 307-410), preferably one Modification in the region of the last 50 residues (i.e., amino acids 361-410). The described mutants were by linker mutagenesis, i. by easy use of existing ones Restriction interfaces in the E. coli serA gene and subsequent insertion of oligonucleotide linkers of a length of 8-14 base pairs.

However, such linker mutagenesis are usually problematic because by the incorporation or deletion of several residues the Structure of the protein is very much changed and so the overall activity or stability of the protein adversely affected becomes. In fact, most of them are described in EP0620853A Mutants a barely detectable activity.

• Peters-Wendisch et al. (2002, Appl. Microbiol. Biotechnol. 60:437-441) beschreiben C-terminale Deletionen der PGD von Corynebacterium glutamicum. Auch hier führen die Deletionen zu teilweise starken Einbußen an Enzymaktivität.
• Die Anmeldung EP0943687A2 beschreibt einen Austausch des Glutaminsäure-Restes an Position 325 der PGD von Brevibacterium flavum. Dieser Rest liegt im Bezug zur Escherichia coli PGD in einem Alignment mit dem Algorithmus GAP des Programms GCG (GCG Wisconsin Package, Genetics Computer Group (GCG) Madison, Wisconsin) bereits in dem variablen C-terminalen Teil des Proteins und korreliert mit dem Asparaginrest 364 des Escherichia coli Proteins. Da diese Modifikation in dem variablen C-terminalen Teil der PGD liegen, sind keine Rückschlüsse auf das Escherichia coli Protein möglich.

Also in coryneform microorganisms, mutageneses have been made on the serA gene that fulfill the goal of reducing the sensitivity of PGD to inhibition by serine:

• Peters-Wendisch et al. (2002, Appl.Microbiol.Biotechnol 60: 437-441) describe C-terminal deletions of PGD of Corynebacterium glutamicum. Again, the deletions lead to some strong losses of enzyme activity.
• The application EP0943687A2 describes an exchange of the glutamic acid residue at position 325 of the PGD of Brevibacterium flavum. This residue is already in the variable C-terminal part of the protein and correlates with the asparagine residue 364 in an alignment with the GAP algorithm of the GCG program (GCG Wisconsin Package, Genetics Computer Group (GCG) Madison, Wisconsin) in relation to Escherichia coli PGD of the Escherichia coli protein. Since these modifications are in the variable C-terminal part of the PGD, no conclusions are possible on the Escherichia coli protein.

Object of the present invention was to variants of PGD of Escherichia coli which have an im Compared to Escherichia coli wild-type PGD decreased sensitivity against inhibition by serine.

The object is achieved by a PGD having an amino acid sequence, which is characterized by being different from the Amino acid sequence of Escherichia coli wild-type PGD (SEQ ID NO: 2) with a methionine as position 1 differs thereby that at position 349 an amino acid except glycine or at position 372 an amino acid except threonine located.

A PGD according to the invention may also have mutations in both Have positions of SEQ ID NO: 1.

The invention further relates to a DNA sequence coding for a PGD according to the invention. This serA allele is different different from the gene of Escherichia coli PGD (serA gene, SEQ ID NO: 1) in that the codon 349 is a natural amino acid Exception of glycine or codon 372 for a natural amino acid coded with the exception of threonine.

A serA allele according to the invention may also have a mutation have two codons mentioned.

In the context of the present invention are as inventive serA alleles include such genes that are analyzed with the algorithm GAP (GCG Wisconsin Package, Genetics Computer Group (GCG) Madison, Wisconsin) a sequence identity greater than 30% if they have any of the above mutations exhibit. Particularly preferred is a sequence identity greater than 70%.

Likewise, proteins with a sequence identity greater than 40 % determined with the algorithm GAP, in the sense of the present To conceive of the invention as proteins derived from E. coli PGD, provided they have a PGD activity and one of the above Have amino acid substitutions. Particularly preferred is a Sequence identity greater than 70%.

Furthermore, genes according to the invention are allelic variants of the serA gene to be understood by deletion, insertion or Substitution of nucleotides from that shown in SEQ ID NO: 1 Derive sequence, with the enzymatic activity of the gene product more than 10% of the activity of the wild-type gene product and a mutation of codon 349 for the Amino acid glycine or codon 372 for the amino acid threonine, or a combination of the two mutations.

PGD variants that have an amino acid substitution at position 349 or at position 372 or a combination thereof, can be generated using standard techniques of molecular biology become. For this purpose, mutations are made in the corresponding codons introduced the serA gene encoding the PGD. Appropriate Methods for introducing mutations at specific positions within a DNA fragment are known.

The starting material for the mutagenesis is preferably the DNA of the E. coli serA gene. The serA gene to be mutated can chromosomally or extrachromosomally encoded. It is preferred However, the serA gene amplified by polymerase chain reaction and cloned into a vector. By applying the aforementioned Mutagenesis methods are one or more nucleotides the DNA sequence is altered so that the encoded PGD undergoes an amino acid exchange at position 349 or 372, wherein position 1 is the starting methionine of SEQ ID NO: 1.

These mutations cause the encoded PGD to diminish one Sensitivity to inhibition by L-serine (= feedback resistance) has. It is particularly advantageous that the PGD variants according to the invention in the absence of L-serine in An activity of more than 10% compared to wild-type PGD, preferably an unchanged activity.

To determine the extent of feedback resistance of a PGD variant according to the invention, any method can be used which allows to determine the activity of the enzyme in the presence of L-serine. For example, the determination of PGD activity can be based on the method described by McKrickick and Pizer (1980, J. Bacteriol 141: 235-245). The enzyme activity is measured by the reverse reaction in an approach containing phosphohydroxypyruvate and NADH / H ⁺ . The reaction is started by enzyme addition and monitored by the decrease in absorbance at 340 nm, which is caused by oxidation of NADH / H ⁺ in a spectrophotometer. The inhibition of PGD activity is tested in the presence of various concentrations of L-serine in the reaction mixture. The catalytic activity of the various PGD variants is determined in the presence and absence of L-serine and used to determine the inhibitor constant K _i . The K _i describes that inhibitor concentration in which the activity is only 50% of the activity determined in the absence of the inhibitor.

PGD enzymes according to the invention can, due to their feedback resistance for the production of amino acids of the phosphoglycerate family or of compounds resulting from the C1 metabolism derive, be used. For this purpose, the inventive serA alleles expressed in a host strain.

The expression of a serA allele according to the invention can be described under Control of the own promoter localized in front of the serA gene or by using other suitable promoter systems, which are known in the art, take place. This can be the corresponding allele, for example, under the control of a such promoter in either one or more copies located on the chromosome of the host organism. The strategies for the integration of genes into the chromosome are state of the art Technology. However, the serA allele to be expressed is preferred cloned into a vector, preferably a plasmid.

The invention therefore also relates to a vector, characterized that he under a serA allelic according to the invention functional control of a promoter.

For cloning the serA alleles according to the invention, vectors which already contain genetic elements (e.g. constitutive or regulatable promoters, terminators), either an ongoing or a controlled, allow inducible expression of the gene coding for the PGD. They are also located on an expression vector preferably other regulatory elements such as ribosomal Binding sites and termination sequences as well as sequences that code for selective markers and / or reporter genes. The expression Such selection marker facilitates identification of transformants. Suitable as selection markers Genes that are resistant to z. B. ampicillin, Tetracycline, chloramphenicol, kanamycin or other antibiotics encode. When the serA allele according to the invention is extrachromosomal should be replicated, the plasmid vector should preferably contain an origin point of replication. Especially preferred are plasmid vectors such as the E. coli vectors pACYC184, pUC18, pQE-70, pBR322, pSC101 and their derivatives. Suitable inducible promoters are, for example the lac, tac, trc, lambda PL, ara or tet promoter or sequences derived therefrom.

• L-Serin (z.B. serB-, serC-, Exportcarrier-Gen wie beschreiben in DE10044831A1)
• N-Acetyl-Serin, O-Acetyl-Serin, Cystin, Cystein oder Cysteinderivaten (z.B. cysE-Allele wie beschrieben in WO97/15673, Efflux-Gene wie beschrieben in EP0885962A1, cysB-Gen wie beschrieben in DE19949579C1, yfiK-Gen wie beschrieben in DE 10232930A)
• L-Tryptophan (z.B. trpE-Allele wie beschrieben in EP0662143A)
• Pantothensäure (z.B. wie beschrieben in WO02061108)

Furthermore, particular preference is given to plasmid vectors which already contain a gene / allele whose use also leads to an overproduction of amino acids of the phosphoglycerate family or of compounds which derive from the C1 metabolism, for example for the production of:

• L-serine (eg serB, serC, export carrier gene as described in DE10044831A1)
• N-acetyl-serine, O-acetyl-serine, cystine, cysteine or cysteine derivatives (eg cysE alleles as described in WO97 / 15673, efflux genes as described in EP0885962A1, cysB gene as described in DE19949579C1, yfiK gene as described in DE 10232930A)
• L-tryptophan (eg trpE alleles as described in EP0662143A)
• Pantothenic acid (eg as described in WO02061108)

Such vectors allow the direct production of inventive Microorganism strains with high production capacity from any microorganism strain, as such Plasmid also the lifting of other restrictions of the Metabolic pathway in a microorganism causes.

By a common transformation method (e.g., electroporation) become the serA allele-containing plasmids according to the invention incorporated in microorganisms and, for example, by antibiotic resistance selected for plasmid-carrying clones.

The invention thus also relates to processes for the preparation a microorganism strain according to the invention, characterized that in a microorganism strain an inventive Vector is introduced.

It is also possible vectors with a serA allele according to the invention in microorganisms, for example, single or more of the above genes / alleles already chromosomally express and already an overproduction of a Have metabolite. In such cases can through the introduction of a serA allele according to the invention the production output be increased again.

Generally, as a host organism for vectors of the invention all organisms suitable for the biosynthetic pathway for amino acids the phosphoglycerate family, recombinant Process are accessible and cultivable by fermentation are. Such microorganisms can be fungi, yeasts or bacteria be. Preference is given to bacteria of the phylogenetic group Eubacteria are used. Particularly preferred are microorganisms the family Enterobacteriaceae and in particular the Species Escherichia coli.

The invention thus further relates to a microorganism strain, for the fermentative production of amino acids of Phosphoglycerate family or their derivatives or compounds, which derive from the C1 metabolism suitable is, characterized in that it is a PGD according to the invention has.

Another object of the invention is the production of Amino acids of the phosphoglycerate family or of compounds, which derive from the C1 metabolism, by cultivation a microorganism strain according to the invention.

For this purpose, the microorganism strain according to the invention, for example cultured in a fermenter in a nutrient medium, the a suitable carbon, and a suitable source of energy, and other additives.

The substances formed during the fermentation such as L-phosphoserine, L-serine, O-acetyl-L-serine, L-cysteine, Glycine, L-tryptophan, 1,2,4-triazol-2-yl-L-alanine, L-methionine or pantothenic acid can subsequently be purified become.

The following examples serve to further explain the Invention. All molecular biological procedures used, like polymerase chain reaction, isolation and purification DNA, modification of DNA by restriction enzymes, Klenow fragment and ligase, transformation, etc. were in the known in the art, described in the literature or recommended by the respective manufacturers way carried out.

Example 1: Cloning of the serA gene

und

The serA gene from Escherichia coli strain W3110 (American Type Culture Collection, ATCC27325) was amplified by the polymerase chain reaction. The oligonucleotides were used as specific primers

and

The resulting DNA fragment was digested with the restriction enzymes NdeI and HindIII digested and the 5 'overhangs with Klenow enzyme refilled. Subsequently, the DNA fragment was using purified by agarose gel electrophoresis and using the GeneClean method isolated (GeneClean Kit BI0101 P.O.Box 2284 La Jolla, California, 92038-2284). The cloning of the thus obtained serA fragment was inserted into the expression vector pQE-70 (Qiagen, Hilden, D). For this, the vector was first with SphI and BamHI cut and the 3 'overhang with Klenow enzyme or the 5 'overhang filled with Klenow enzyme. Subsequently, the vector fragment was purified and washed with ligated to the serA fragment. The resulting vector carries the Designation pFL209. After verification of the construct by sequencing the Escherichia coli strain JM109 (Stratagene, Amsterdam, NL) and corresponding Transformants selected with ampicillin. The bacterial strain Escherichia coli JM109 / pFL209 was used in the DSMZ (German Collection for microorganisms and cell cultures GmbH, D-38142 Braunschweig) under the number DSM 15628 according to the Budapest Treaty deposited.

Example 2: Site-directed mutagenesis of the serA gene

und

verwendet.Site-directed mutagenesis at codons 349 and 372 of the serA gene was performed by an inverse polymerase chain reaction. The template used was the vector pFL209 described in Example 1. For the mutagenesis of codon 349, the primers were

and

used.

und

verwendet.The resulting PCR product was circularized by ligation and transformed into E. coli strain JM109. Sequencing finally determined the mutation at codon 349 and checked the correctness of the remaining sequence.
In principle the same procedure was followed for the mutagenesis of codon 372, but the primers became

and

used.

Example 3: Determination of PGD Activity and Inhibitor Constant K _i

For the determination of PGD enzyme activities and the influence of L-serine on the activity, 100 ml LB medium (10 g / l tryptone, 5 g / l yeast extract, 10 g / l NaCl), which additionally contained 100 mg / l ampicillin , inoculated with a 2 ml overnight culture of the strains with the plasmid-coded serA alleles and incubated at 30 ° C and 150 rpm in a shaker. At 1.0 optical density, serA expression was induced by the addition of 0.4 mM isopropyl-β-thiogalactoside, and the culture was incubated for a further 3 hours. The cells were then harvested by centrifugation, washed and resuspended in 2 ml of buffer (100 mM K-phosphate pH 7.0, 10 mM MgCl ₂ , 1 mM dithiothreitol). Cell disruption was performed using a French Press (Spectronic Instruments, Inc., Rochester, NY, USA) at a pressure of 18,000 psi. The crude extracts were clarified by centrifugation at 30,000 g and PGD activity was determined by the test of McKrickick and Pizer (1980, J. Bacteriol 141: 235-245).
The following tables show the PGD activity of different mutants, as well as the corresponding inhibitor constants K _i . Mutations at codon 349 allele mutation Activity [units / mg] Ki [mM] serA wildtype 0.05 <0.1 serA40 G349D 0.05 25 serA45 G349I 0.05 5 serA46 G349M 0.05 1 serA47 G349E 0.05 20 serA49 G349P 0.05 6 serA410 G349S 0.04 2 serA411 G349T 0.04 3 serA412 G349V 0.05 5 serA413 G349L 0.05 5 serA414 G349A 0.05 1 serA415 G349K 0.03 15 serA416 G349R 0.04 15 serA417 G349W 0.02 8th serA418 G349Y 0.05 6 serA419 G349F 0.05 10 serA420 G349H 0.05 10 serA421 G349N 0.05 15 serA422 G349Q 0.05 15 serA45 G349C 0.04 5 Mutations at codon 372 allele mutation Activity [units / mg] Ki [mM] serA wildtype 0.05 <0.1 serA20 T372I 0.05 40 Ser A21 T372D 0.05 120 serA211 T372Y 0.05 35 serA219 T372G 0.05 8th serA220 T372S 0.05 1 serA223 T372E 0.05 150 serA229 T372R 0.05 120 serA234 T372K 0.05 110 serA206 T372P 0.05 120 serA208 T372H 0.05 80 serA210 T372W 0.04 60 serA212 T372F 0.05 60 serA214 T372A 0.04 10 serA218 T372N 0.05 100 serA221 T372Q 0.05 100 serA222 T372V 0.05 40 serA226 T372L 0.04 40 serA228 T372M 0.03 60 serA231 T372C 0.02 3

Example 4: Combination of mutations of serA20 and alleles serA40

A combination of codons 349 and 372 mutations should show whether the exchanges have a synergistic effect on feedback resistance. For this purpose, a unique HindII restriction site between the two mutation sites was used. Thus, HindIII-HindIII restriction of the serA20 allele vector isolated a 183 bp fragment corresponding to the 3 'end of the serA gene and containing the mutation at codon 372. This fragment was cloned into a similarly Hindllam-BamHI digested vector with the serA40 allele to give a clone which is a double mutant. The following table shows the associated enzyme data. Mutations at codons 349 and 372 allele mutation Activity [units / mg] Ki [mM] serA wildtype 0.05 <0.1 serA2040 G349D, T372I 0.05 120

Claims (10)

3-Phosphoglycerate dehydrogenase (PGD) having a reduced susceptibility to serine inhibition with respect to Escherichia coli wild-type PGD having an amino acid sequence characterized by being different from the amino acid sequence of Escherichia coli wild-type PGD (SEQ. ID NO: 2) differs in that at position 349 there is an amino acid with the exception of glycine or at position 372 an amino acid with the exception of threonine. A 3-phosphoglycerate dehydrogenase according to claim 1, characterized in that at position 349 there is an amino acid with the exception of glycine and at position 372 an amino acid with the exception of threonine. A serA allele encoding a PGD according to claim 1, characterized in that it differs from the gene of Escherichia coli PGD (serA gene, SEQ ID NO: 1) in that the codon 349 for a natural amino acid with the exception of glycine or the Codon 372 encoded for a natural amino acid except threonine. A serA allele coding for a PGD according to claim 2, characterized in that it differs from the gene of Escherichia coli PGD (serA gene, SEQ ID NO: 1) in that the codon 349 for a natural amino acid with the exception of glycine and the Codon 372 encoded for a natural amino acid except threonine. The serA allele according to claim 3 or 4, characterized in that the enzymatic activity of the gene product is maintained so that it corresponds to more than 10% of the activity of the wild-type gene product and a mutation of the codon 349 for the amino acid glycine and the codon 372 for the Amino acid threonine, or a combination of the two mutations is present. Vector, characterized in that it contains a serA allele according to claim 3, 4 or 5 under the functional control of a promoter. Process for the preparation of a microorganism strain, characterized in that a vector according to claim 6 is introduced into a microorganism strain. Microorganism strain which is suitable for the fermentative production of amino acids of the phosphoglycerate family or their derivatives or of compounds deriving from the C1 metabolism, characterized in that it has a PGD according to claim 1 or 2. Process for the preparation of amino acids of the phosphoglycerate family or of compounds which result from the C1 Derive metabolism by cultivating a microorganism strain according to claim 8. Use of a PGD according to claim 1 or 2 for the preparation of amino acids of the phosphoglycerate family or of Compounds derived from C1 metabolism.